Pharmacokinetic neuroimaging to study the dose-related brain kinetics and target engagement of buprenorphine in vivo Sylvain Auvity, Sébastien Goutal, Fabien Caillé, Dominique Vodovar, Alain Pruvost, Catriona Wimberley, Claire Leroy, Matteo Tonietto, Michel Bottlaender, Nicolas Tournier

To cite this version:

Sylvain Auvity, Sébastien Goutal, Fabien Caillé, Dominique Vodovar, Alain Pruvost, et al.. Pharma- cokinetic neuroimaging to study the dose-related brain kinetics and target engagement of buprenor- phine in vivo. Neuropsychopharmacology, Nature Publishing Group, 2021, 46 (6), pp.1220-1228. ￿10.1038/s41386-021-00976-w￿. ￿cea-03215261￿

HAL Id: cea-03215261 https://hal-cea.archives-ouvertes.fr/cea-03215261 Submitted on 3 May 2021

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Pharmacokinetic neuroimaging to study the dose-related brain kinetics and target 2 engagement of buprenorphine in vivo

3 Sylvain AUVITY1,2 (PhD, PharmD), Sébastien GOUTAL1,3 (MSc), Fabien CAILLÉ1,4 (PhD), 4 Dominique VODOVAR1,2 (PhD, MD), Alain PRUVOST5 (PhD), Catriona WIMBERLEY1,6 5 (PhD), Claire LEROY1,4 (PhD), Matteo TONIETTO1,4 (PhD), Michel BOTTLAENDER1,4 (PhD, 6 MD), Nicolas TOURNIER1,4 (PhD, PharmD)

7

8 1. CEA/DRF/JOLIOT/Service Hospitalier Frédéric Joliot, 91401 ORSAY France 9 2. UMR-S 1144, Université de Paris, 75005 PARIS France 10 3. MIRCen, CEA/IBFJ/DRF-JACOB/LMN, UMR CEA CNRS 9199, Université Paris 11 Saclay, Fontenay-aux-Roses, France 12 4. Université Paris-Saclay, Inserm, CNRS, CEA, Laboratoire d'Imagerie Biomédicale 13 Multimodale Paris-Saclay, 91401 Orsay 14 5. Service de Pharmacologie et d'Immunoanalyse (SPI), Plateforme Smart-MS, CEA, 15 INRA, Université Paris-Saclay, 91191, Gif-sur-Yvette, France 16 6. Edinburgh Imaging, Queen's Medical Research Institute, University of Edinburgh, 17 Edinburgh, UK 18

19 Corresponding author: Nicolas Tournier (PhD, PharmD)

20 [email protected]

21 Université Paris-Saclay, CEA, CNRS, Inserm, BioMaps,

22 Service Hospitalier Frédéric Joliot,

23 4 place du général Leclerc,

24 91401 ORSAY France

25 Phone +33 1 69 86 77 12

26 Fax + 33 1 69 86 77 86

27

28

29

30

31

1 1

2 Abstract (250 words)

3 A wide range of buprenorphine doses are used for either pain management or maintenance 4 therapy in opioid addiction. The complex in vitro profile of buprenorphine, with affinity for µ-, 5 δ- and κ-opioid receptors (OR), makes it difficult to predict its dose-related 6 neuropharmacology in vivo. In rats, microPET imaging and pretreatment by OR antagonists 7 were performed to assess the binding of radiolabeled buprenorphine (microdose 11C- 8 buprenorphine) to OR subtypes in vivo (n=4 per condition). The µ-selective antagonist 9 naloxonazine (10 mg/kg) and the non-selective OR-antagonist naloxone (1 mg/kg) blocked 10 the binding of 11C-buprenorphine while pretreatment by the δ-selective (naltrindole, 3 mg/kg) 11 or the κ-selective antagonist (norbinaltorphimine, 10 mg/kg) did not. In four macaques, PET 12 imaging and kinetic modeling enabled description of the regional brain kinetics of 11C- 13 buprenorphine, co-injected with increasing doses of unlabeled buprenorphine. No saturation 14 of the brain penetration of buprenorphine was observed for doses up to 0.11 mg/kg. Regional 15 differences in buprenorphine-associated receptor occupancy were observed. Analgesic 16 doses of buprenorphine (0.003 and 0.006 mg/kg) respectively occupied 20% and 49% of 17 receptors in the thalamus while saturating the low but significant binding observed in 18 cerebellum and occipital cortex. Occupancy >90% was achieved in most brain regions with 19 plasma concentrations >7 µg/L. PET data obtained after co-injection of an analgesic dose of 20 buprenorphine (0.003 mg/kg) predicted the binding potential of microdose 11C- 21 buprenorphine. This strategy could be further combined with pharmacodynamic exploration 22 or pharmacological MRI to investigate the neuropharmacokinetics and neuroreceptor 23 correlate, at least at µ-OR, of the acute effects of buprenorphine in humans.

24

2 1 Introduction

2 The thebaine derivative buprenorphine is a semi-synthetic opioid of the phenanthrene family 3 [1]. Low-dose buprenorphine offers potent analgesia for the treatment of moderate to severe 4 pain in patients. Compared with other opioids, buprenorphine benefits from a unique safety 5 profile, with limited risk for respiratory depression and overdose. High-dose buprenorphine is 6 therefore approved for addiction maintenance therapy in the management of opioid use 7 disorders with a growing interest in the context of the current opioid crisis [1–3].

8 In vitro, buprenorphine is one of the most affine ligand of the human µ-opioid receptor (µ-OR,

9 Ki=0.9 nM) and was compared with other opioid such as naloxone (Ki=14 nM), morphine

10 (Ki=74 nM) or oxycodone (Ki=780 nM) in the same conditions [4,5]. In vitro, buprenorphine is 11 also far more potent than morphine at stimulating µ-OR, with half-maximal effective

12 concentration (EC50)<0.1 nM and 130 nM for buprenorphine and morphine, respectively, 13 although buprenorphine shows lower maximum efficacy than morphine in mediating µ-OR 14 coupling [4]. Buprenorphine was therefore classified as a highly potent but partial agonist of 15 µ-OR [1,6]. Buprenorphine shows a slow dissociation rate from µ-OR, assumed to account 16 for prolonged occupancy and duration of action in vivo [7,8]. Buprenorphine is also described 17 as antagonist of κ-OR and δ-OR, and agonist of nociceptin/ORL-1 receptors [9,10]. It is 18 therefore difficult to predict the in vivo dynamics of the interaction of buprenorphine with its 19 CNS targets from this complex in vitro profile.

20 There are still discrepancies in the description of the neuropharmacology of buprenorphine 21 [9,11]. In vivo, buprenorphine benefits from limited respiratory effects at high doses [12]. A 22 “ceiling” or “inverted U-shape” analgesic dose-response has been described in animals [13]. 23 However, in patients, buprenorphine shows a dose-dependent analgesic effect similar than 24 that of full agonists [11]. Peripheral pharmacokinetics of buprenorphine is well established in 25 humans [14]. Norbuprenorphine (N-dealkyl-buprenorphine) is the predominant metabolite 26 and shows negligible blood-brain barrier (BBB) penetration compared with buprenorphine 27 [15]. Its relatively short elimination half-life of ~3h contrasts with its prolonged duration of 28 action [1,11,14], suggesting particular brain kinetics.

29 Pharmacological Positron Emission Tomography (PET) imaging uses target-specific 30 radioligands to capture the target engagement associated with one controlled plasma level of 31 the investigated drug [16]. The µ-OR-selective radioligand 11C-carfentanil [17] was used in 32 healthy volunteers and heroin-dependent patients to estimate the extent and duration of µ- 33 OR occupancy associated with high-doses of buprenorphine (2-16 mg, sublingual route) [18– 34 20]. Data regarding receptor occupancy associated with acute administration of analgesic 35 doses of buprenorphine (0.3-0.6 mg) are still lacking. Interestingly, isotopic radiolabeling of

3 1 buprenorphine is feasible [21]. This provides a unique opportunity for direct determination of 2 the brain kinetics of buprenorphine, at its site of action, a strategy named pharmacokinetic 3 imaging [22]. Moreover, pharmacological doses of buprenorphine, instead of microdose 4 usually encountered in PET studies, can be safely used to mimic the clinical situation in 5 terms of pharmacokinetics and pharmacodynamics.

6 In the present study, pharmacokinetic imaging using 11C-buprenorphine was performed to 7 explore the neuropharmacology of buprenorphine in vivo. Blocking experiments were 8 performed to address the binding of 11C-buprenorphine to µ-, δ- and κ-OR in rats. 11C- 9 buprenorphine PET imaging was then performed in macaques to assess the regional 10 neuropharmacokinetics and receptor occupancy of buprenorphine associated with a wide 11 range of buprenorphine doses, that covers its clinical use in both analgesia and addiction 12 maintenance.

13

4 1 Material and Methods

2 1. Chemicals

3 Buprenorphine hydrochloride for i.v. injection (0.3 mg/mL) was obtained from Axience 4 (Pantin, France). Naloxone hydrochloride for i.v. injection (0.4 mg/mL) was obtained from 5 Aguettant (Lyon, France). Naloxonazine and norbinaltorphimine were obtained from Sigma- 6 Aldrich (Saint-Quentin Fallavier, France) and naltrindole from Tocris (Noyal-Chatillon sur 7 Sèche, France). Ketamine was obtained from Virbac (Caros, France). Propofol was 8 purchased from Fresenius laboratory (Sèvres, France). Isoflurane was obtained from Abbvie 9 (Rungis, France). 11C-Buprenorphine was synthesized in-house according to the method 10 described by Lever et al. [21] with slight modifications (see supplemental material).

11 2. Animals

12 All animal use procedures were in accordance with the recommendations of the European 13 Community for the care and use of laboratory animals (2010/63/UE) and the French National 14 Committees (French Decret 2013-118). Experimental protocols were validated by a local 15 ethics committee for animal use (CETEA/A15-002 and A18-065) and approved by the french 16 government. Rodent experiments were conducted in male Sprague-Dawley rats (224±43g). 17 Each rat underwent a single PET experiment. Four adult male rhesus macaques (Macaca 18 Mulatta; 8.4±3.4 kg in weight during the study) were obtained from Silabe (Simian Laboratory 19 Europe, France). A minimum interval of 2 weeks was respected between two scans in the 20 same individual.

21 3. Binding of 11C-buprenorphine to OR subtypes in rats

22 MicroPET imaging

23 11C-buprenorphine brain PET acquisitions were performed using an Inveon microPET 24 scanner (Siemens Medical Solutions, France). Anesthesia was induced and thereafter

25 maintained using 3% and 1.5–2.5% isoflurane in O2, respectively. A catheter was inserted in 26 a lateral caudal vein for intravenous (i.v.) injection of tested OR-antagonists when necessary. 27 Microdose 11C-buprenorphine (34±7 MBq, 3±2 µg, mean molar activity at time of injection -1 28 MAinj=8.2±4.4 GBq.µmol ) was then injected in the same catheter.

29 Pharmacological challenges

30 Blocking experiments were performed to investigate the binding of 11C-buprenorphine to 31 different OR subtypes in the living brain (n=4 per condition). PET acquisitions were 32 performed without or after previously reported blocking conditions using the non-selective 33 OR antagonist naloxone (1 mg/kg i.v., 5 min before 11C-buprenorphine injection) [23], the

5 1 selective µ-OR antagonist naloxonazine (10 mg/kg i.v., 5 min before 11C-buprenorphine 2 injection) [24], the selective κ-OR antagonist norbinaltorphimine (10 mg/kg intraperitoneal 3 injection, 30 min before 11C-buprenorphine injection) [25] and the selective δ-OR antagonist 4 natrindole (3 mg/kg i.v., 5 min before 11C-buprenorphine injection) [26].

5 Data analysis

6 MicroPET images were reconstructed as previously described [27]. Late brain PET images 7 acquired 40-60min after 11C-buprenorphine injection were coregistered to the Schiffer rat 8 brain template using PMOD software V3.9 (PMOD Technologies, Zürich, Switzerland). 9 Cerebellum was shown devoid of µ- and δ-OR with limited expression of κ-OR in rats [28]. 10 Regional uptake ratios (region/cerebellum) were calculated in baseline and blocking 11 conditions to take any change in peripheral pharmacokinetics of 11C-buprenorphine into 12 account.

13 4. Target engagement of buprenorphine in macaques

14 Co-injection study

15 Further PET experiments were performed in macaques to allow for accurate arterial blood 16 sampling during PET acquisition. First, microdose 11C-buprenorphine was i.v. injected, 17 followed by a 90 min brain PET acquisition. Then, the dose-dependent receptor occupancy 18 associated with therapeutic doses was addressed using a co-injection strategy. Increasing 19 doses of unlabeled buprenorphine (0; 0.003; 0.006; 0.03; 0.06 and 0.011 mg/kg, equivalent 20 to human doses ranging from 0 to 8 mg/70 kg, n=4 per dose) were mixed in the syringe 21 containing microdose 11C-buprenorphine (8.34±3.85 µg). The preparation was i.v. injected at 22 the start of dynamic PET acquisition (90 min).

23 Acquisition procedure

24 First, each monkey underwent an anatomical T1-weighted brain MR scan using an Achieva 25 1.5T scanner (Philips Healthcare, Suresnes, France) under ketamine anesthesia 26 (intramuscular injection, i.m.). PET acquisitions were performed on a HR+ Tomograph 27 (Siemens Healthcare, Knoxville, TN, USA) in anesthetized macaques as previously 28 described [29]. Briefly, the macaque received ketamine (10 mg/kg, i.m.) to induce 29 anesthesia. After intubation in supine position, venous catheters were inserted for radiotracer 30 injection (sural vein), propofol infusion (sural vein) and drug injection for the displacement 31 experiments (brachial vein). Another catheter was inserted into the femoral artery for arterial 32 blood sampling. Macaques were positioned under the camera before administration of a 2 33 mL i.v. bolus of propofol followed by a 1 mL/kg/h i.v. infusion under oxygen ventilation. 11 34 Macaques were i.v. injected with microdose C-buprenorphine (241±42 MBq, MAinj=13.5±5.1

6 1 GBq/µmol). Increasing doses of unlabeled buprenorphine were added to 11C-buprenorphine 2 microdose for the co-injection study. Physiological monitoring, including heart rate, oxygen

3 saturation (SpO2), respiratory rate, and end-tidal CO2, was performed throughout the duration 4 of the PET scan.

5 Imaging data reconstruction and segmentation

6 A post-reconstruction method was performed on dynamic PET image for noise reduction and 7 improved spatial resolution (see supplemental material) [30,31]. PET data were then 8 analyzed using PMOD software. PET images were coregistered to corresponding T1-weigted 9 MR images for each macaque. A macaque T1-weighted MR template [32] was normalized 10 onto individual MR images. Transformation matrices were then applied to the segmentation 11 obtained from the template to generate time-activity curves in 12 selected brain structures.

12 5. Arterial Input Function and Metabolism

13 During PET acquisition, arterial blood samples (500 µL) were withdrawn at selected times 14 after radiotracer injection. Samples were centrifuged (5 min; 2,054g; 4°C) and the 15 supernatant (200 µL) was gamma-counted for total plasma radioactivity. Additional plasma 16 samples were withdrawn at 0; 5; 10; 15; 30; 60 and 90 min to measure both i) the percentage 17 of parent (unmetabolized) 11C-buprenorphine using radio-HPLC and a state-of-the-art 18 methodology [33] and ii) the total concentration of buprenorphine in plasma using mass 19 spectrometry, after radioactive decay. The fraction of parent 11C-buprenorphine in each 20 sample was used to generate the metabolite-corrected arterial input function for 21 pharmacokinetic modeling of each PET experiment (see supplemental material, Fig. S1).

22 6. Pharmacokinetic modeling

23 Kinetics of radioactivity in the brain and in plasma samples were decay-corrected and 24 expressed as the percentage of injected dose of radioactivity per volume (%ID.cm-3). Kinetic 25 modeling was performed considering the metabolite-corrected arterial input function. The 11 26 initial transfer rate of C-buprenorphine from plasma into the brain (K1) was estimated using 27 the graphical plot analysis, as previously described [27] (see supplemental material, Fig. S2). 11 -3 28 The brain distribution of C-buprenorphine (VT; mL.cm ) was estimated using the Logan plot

29 graphical method [34]. Parametric images (VT unit) were generated using PMOD to display 30 the regional brain distribution of 11C-buprenorphine in tested conditions (Fig. 2).

31 Brain data obtained with 11C-buprenorphine co-injected with the maximal dose of unlabeled 32 buprenorphine (0.11 mg/kg) were used to estimate the non-specific binding of 11C- 33 buprenorphine (saturation scan) and define the non-displaceable volume of distribution

34 (VND,saturation) in each region for each animal. Regional VND,saturation were compared with

7 1 graphically estimated VND,graphical (Table 1 and supplemental material, Fig. S3). For each scan, 2 the specific binding of 11C-buprenorphine in each brain region was estimated as the binding

3 potential relative to plasma (BPp) [35] with:

4 BPp = VT - VND,saturation

5 BPp estimated in microdose scans (BPp,microdose) was used to estimate the receptor occupancy 6 associated with each pharmacological dose of unlabeled buprenorphine as follow [35]:

7 Receptor Occupancy (%) = (BPp,microdose - BPp,dose) / BPp,microdose x 100

8 The occipital cortex showed the lowest PET signal and was used as pseudo-reference tissue 9 to estimate the regional binding of 11C-buprenorphine without arterial blood sampling (DVR, 10 Logan reference method) [36]. Occipital cortex commonly serves as a reference region for

11 quantification of PET radioligands targeting µ-OR in humans and monkeys [18,19,37,38]. BPp 12 and DVR are unitless values.

13 For each region and for each scan, the receptor occupancy of 11C-buprenorphine was fitted 14 to the corresponding plasma concentration of buprenorphine, measured from 60 to 90 min 15 post-injection. Occupancy associated with plasma concentrations of buprenorphine obtained 16 with the 0.11 mg/kg dose were set to 100%. A non-linear fit model of saturation with one 17 binding site was used to estimate i) the plasma concentration of buprenorphine associated

18 with regional half-maximum receptor occupancy (EC50) and ii) the receptor occupancy 19 associated with selected plasma levels of buprenorphine (GraphPad Prism software V7.0, 20 San Diego, CA, USA) (see supplemental material, Fig S4, Table 1).

21 7. Displacement experiments in nonhuman primates

22 Additional experiments were performed to address the reversibility of 11C-buprenorphine 23 binding to CNS targets. Displacement experiments were performed in 3 macaques and 24 consisted in the injection of unlabeled buprenorphine (0.03 mg/kg) or naloxone (0.22 mg/kg), 25 30 min after 11C-buprenorphine injection. The selected dose of naloxone is the maximum 26 recommended dose as an antidote against opioid overdose in humans [39]. Methods and 27 results of displacement experiments are reported as supplemental material (Fig. S5).

28 8. Statistical analysis

29 Statistical comparison between conditions was performed using GraphPad Prism. Outcome 30 parameters were compared using a 2-way ANOVA and the Tukey’s post-hoc test. A result 31 was deemed significant when a 2-tailed p value was less than 0.05.

32

8 1 Results

2 1. Binding of 11C-buprenorphine to OR subtypes in rats

3 Baseline brain distribution of 11C-buprenorphine showed high PET signal in the thalamus, 4 striatum and hypothalamus with the lowest PET signal in the cerebellum. Significant 5 differences in uptake ratios were observed across brain regions (p<0.001, Fig. 1). Blocking 6 experiments using the non-selective OR antagonist naloxone, used as positive control, 7 significantly decreased 11C-buprenorphine binding in most brain regions, reaching similar 8 levels than in the cerebellum (p>0.05). Blocking by the selective µ-OR antagonist 9 naloxonazine produced similar effects than naloxone. The binding of 11C-buprenorphine was 10 not significantly decreased by selective blocking of κ-OR (norbinaltorphimine) and δ-OR 11 (naltrindole) (Fig. 1).

12 2. Co-injection study in macaques

13 PET images obtained in monkeys injected with microdose 11C-buprenorphine are shown in 14 Fig. S6. The PET signal slowly accumulated in OR-rich regions such as the putamen, 15 caudate and thalamus. The maximum brain concentration was 0.0255±0.0052 %ID.cm-3 at

16 tmax=22.5 min. Regions with minimal OR expression (cerebellum and occipital cortex)

17 reached their maximum concentration earlier (tmax=6.6 min) with faster decrease of the 18 radioactivity (Fig. S5).

19 Then, 11C-buprenorphine was co-injected with increasing doses of unlabeled buprenorphine 20 up to 0.11 mg/kg (Fig. 2). Buprenorphine doses were well tolerated and no change in 21 physiological parameters was observed. Selected doses of unlabeled buprenorphine did not 22 impact the metabolism and plasma kinetics of 11C-buprenorphine with no difference in 23 plasma exposure (p>0.05, Fig S1). This suggests a linear pharmacokinetics for 24 buprenorphine in plasma within the tested dose range. The plasma concentrations of 25 unlabeled buprenorphine estimated from 60 to 90 min ranged from 0.10±0.08 µg/L 26 (microdose condition) to 11.56±2.94 µg/L (0.11 mg/kg condition) and were significantly 27 correlated with injected dose (Fig. S7).

28 Kinetic modeling was performed to estimate 11C-buprenorphine distribution to brain regions in 29 the presence of increasing doses of unlabeled buprenorphine. Co-injection of unlabeled 11 30 buprenorphine up to 0.11 mg/kg did not impact the K1 of C-buprenorphine from plasma into

31 the brain (p>0.05, Fig. S3). There was no difference in K1 between brain regions (p>0.05). 11 32 Parametric mapping of VT obtained using microdose C-buprenorphine showed significant

33 differences in regional VT between OR-rich brain regions such as the thalamus, striatal and

9 1 cortical regions and OR-poor regions such as the cerebellum and occipital cortex (p<0.01, 2 Fig. 2).

3 The lowest dose of unlabeled buprenorphine (0.003 mg/kg) was sufficient to saturate 11C- 4 buprenorphine binding in the cerebellum and occipital cortex. OR-rich regions showed a

5 dose-dependent decrease in VT, with a maximal 2.5-fold decrease observed in the putamen 6 obtained using the 0.06 mg/kg dose (Fig. 2). Higher dose (0.11 mg/kg) did not further

7 decrease VT, suggesting complete saturation of buprenorphine brain targets at the 0.06 8 mg/kg dose. 11C-buprenorphine-associated radioactivity at doses higher than 0.06 mg/kg 9 predominantly reflected the non-specific binding of 11C-buprenorphine and there was no

10 difference in regional VT across brain regions at either 0.06 mg/kg or 0.11 mg/kg (p>0.05). 2 11 We found a strong correlation between VND,saturation and VND,graphical (p<0.001, R = 0.997, Table

12 1, Fig. S3). VT estimated at 0.11 mg/kg therefore provides a good estimate of the regional 11 13 non-displaceable volume of distribution (VND) of C-buprenorphine for each individual (Fig. 14 2).

15 In Figure 3, regional VT estimated for each dose of unlabeled buprenorphine was plotted to

16 microdose VT according to the VT,dose=f(VT,microdose) equation. The lowest dose of unlabeled 17 buprenorphine (0.003 mg/kg) did not impact the slope of the equation which remained ~1.0, 18 suggesting negligible occupancy in most brain regions. Higher doses of unlabeled

19 buprenorphine did not further decrease VT in the occipital cortex and cerebellum but induced 20 a dose-dependent decrease in the slope of the equation. Deviation of the slope from zero 21 was not significant (p>0.05) for doses of buprenorphine ≥0.06 mg/kg, suggesting total 22 occupancy (Fig. 3) [40].

11 23 There was a strong correlation between regional VT and BPp obtained with C- 2 24 buprenorphine (R =0.98, p<0.001, Fig. 4). Thus, microdose VT accurately predicted the total 11 25 specific binding of C-buprenorphine. VT and BPp values estimated with kinetic modeling 26 were used as a gold-standard to test the reliability of the Logan reference method using the

27 occipital cortex as a pseudo-reference region [36] (Fig. 4). DVRmicrodose and DVR0.003mg/kg were 28 not significantly different (p>0.05, paired t-test, Table 1, Fig. 4), suggesting similar relative

29 binding across brain regions. DVRmicrodose or DVR0.003mg/kg correlated with VT,microdose (p<0.001; 30 R2=0.43 and 0.54, respectively, data not shown). Better correlation was found between 2 31 DVRmicrodose or DVR0.003mg/kg and BPp,microdose (p<0.001; R =0.64 and 0.66, respectively). This 32 suggests that DVR estimated using 11C-buprenorphine/buprenorphine at either microdose or 11 33 0.003 mg/kg predicted the regional BPp of microdose C-buprenorphine (Fig. 4).

34 Plasma concentrations of buprenorphine associated with analgesic doses of buprenorphine 35 (0.003 mg/kg and 0.006 mg/kg) were 0.29±0.04 µg/L and 0.66±0.22 µg/L, respectively (Fig.

10 1 S7). In the thalamus, corresponding receptor occupancy was 20% and 49%, respectively. 2 Regions with the lowest specific binding (cerebellum and occipital cortex) were fully occupied

3 at the lowest analgesic dose. Thus, poor fit and estimation of EC50 were obtained in these

4 regions (Table 1, Fig. S4). In most OR-rich regions, receptor occupancy >90% was achieved 5 with plasma concentrations of buprenorphine >7 µg/L (Table 1, Fig. S4). Regional receptor 6 occupancies associated with a range of plasma concentrations of buprenorphine were

7 estimated (Table 1). Regional differences in receptor occupancy and EC50 could be noticed.

8

9

11 1 Discussion

2 PET imaging studies using µ-OR-targeting radioligands are classically used for estimation of 3 the interaction of opioids with µ-OR, with limited information on brain kinetics of investigated 4 compounds [41]. Pharmacokinetic PET studies using radiolabeled analogues of drugs are 5 increasingly used for direct determination of their BBB penetration or brain delivery [22]. This 6 microdose strategy does not however provide information regarding pharmacodynamics, as 7 compared with behavioral investigation or pharmacological MRI (phMRI) [42,43]. We used 8 complementary pharmacokinetic neuroimaging approaches using 11C-buprenorphine to 9 directly assess its binding to OR subtypes in vivo, as well as the dose-related brain kinetics 10 and target engagement associated with clinically relevant doses of unlabeled buprenorphine.

11 11C-buprenorphine PET signal in brain regions depends on its non-specific binding, its affinity 12 for OR subtypes, their regional availability and corresponding association/dissociation 13 kinetics. Binding of buprenorphine to µ-OR, κ-OR and δ-OR has been compared in the same

14 in vitro conditions. Respective Ki of buprenorphine for µ-, κ- and δ-OR was 0.08, 0.44 and 15 0.82 nM (monkey), 0.08, 0.11 and 0.42 nM (rat) and 12.4, 108 and 154 nM (human).

16 Buprenorphine showed much lower affinity for ORL-1 (Ki = 285 nM in rats) [9,44]. Our 17 blocking experiments in rats suggest that the specific binding of 11C-buprenorphine 18 predominantly reflects its interaction with µ-OR rather than κ- or δ-OR. This is consistent with 19 previous ex vivo data showing a single predominant high affinity binding site for 3H- 20 buprenorphine in rat brain lysate, leading to linear Scatchard plot in saturation experiments 21 [45]. Frost et al. compared the regional binding specificity of the non-selective OR antagonist 22 11C-diprenorphine and µ-OR-selective agonist 11C-carfentanil in humans using the thalamus, 23 a region with known predominance of µ-OR, as a normalization region [46]. Using the same 24 method with our macaque data, the regional binding of 11C-buprenorphine obtained using 25 either microdose or co-injection of 0.003 mg/kg of unlabeled buprenorphine fits the regional 26 distribution of 11C-carfentanil rather than that of 11C-diprenorphine (Fig. S8, Table S1). In 27 pharmacotherapy, the affinity for κ- and δ-OR was shown to account for the 28 pharmacodynamics of high-dose buprenorphine [9,44]. However, from a molecular imaging 29 perspective, only the µ-OR component of the neuropharmacology of buprenorphine can be 30 estimated using 11C-buprenorphine PET imaging.

31 Modest but significant specific binding of 11C-buprenorphine was observed in the cerebellum 32 and occipital cortex in macaques, in both our co-injection and displacement experiments 33 (Fig. S5). Data regarding the expression of OR in these brain regions in monkeys are scarce 34 [47]. Although species differences in OR expression may exist, it was reported a low but 35 significant local expression of µ-, κ- but not δ-OR in the human cerebellum [48,49]. In the

12 1 human occipital cortex, expression of κ-, δ- but not µ-OR has been detected [50,51]. In other 2 regions with known µ-OR expression, unlabeled buprenorphine dose-dependently decreased 11 11 3 C-buprenorphine VT (Fig. 2 and 3). No saturation of the BBB penetration of C- 4 buprenorphine was observed for doses up to 0.11 mg/kg (Fig. S2). Full saturation of 5 neuroreceptors, achieved with the highest doses of buprenorphine, revealed the 6 homogenous mapping of the non-specific binding of 11C-buprenorphine (Fig. 2). Thus, 7 quantitative data regarding total specific binding potential of 11C-buprenorphine to CNS

8 targets (BPp) could be derived (Table 1).

9 Estimation of the dose-related receptor occupancy by buprenorphine using a target-specific 10 radioligand such as 11C-carfentanil or 11C-diprenorphine may depend on the affinity of the 11 selected probe for investigated OR [52]. Direct saturation experiments with 11C- 12 buprenorphine/buprenorphine therefore provide a unique in vivo translation of in vitro binding 13 experiments [7]. Plasma levels associated with analgesic doses of buprenorphine (0.003 and 14 0.006 mg/kg) ranged from 0.29±0.04 to 0.66±0.22 µg/L, consistent with clinical 15 pharmacokinetic data in patients [14]. Corresponding plasma levels of buprenorphine 16 occupied <50% of the total binding in µ-OR-rich regions such as the thalamus. This suggests 17 that partial occupancy of µ-OR is sufficient to achieve effective analgesia, which may also 18 involve action on κ-OR and nociceptin/ORL-1 at the spinal level [53].

19 Buprenorphine for addiction maintenance is administered via sublingual route (bioavailability 20 ~70%) [54]. Buprenorphine plasma levels associated with the lowest dose used for addiction 21 maintenance (0.03 mg/kg), administered i.v., still partially occupied µ-OR. Full receptor 22 occupancy was achieved with doses ≥0.06 mg/kg. It was suggested that >50% of µ-OR 23 occupancy is required to ensure suppression of withdrawal syndrome. Moreover, µ-OR 24 occupancy >80% is assumed to protect against opioid overdose syndrome induced by 25 massive and unintended intake [20]. Our macaque data suggest that plasma concentrations 26 >7 µg/L have to be maintained to ensure >90% occupancy of OR by buprenorphine in the 27 striatum. In patients, higher doses of buprenorphine may thus essentially maintain plasma 28 concentration over the targeted threshold to ensure sustained and effective maintenance 29 therapy [55]. This observation is consistent with previous 11C-carfentanil PET data obtained 30 in heroin-dependent subjects showing that total µ-OR occupancy is prolonged by increasing 31 the doses of buprenorphine [19].

32 Compared with other opioids, buprenorphine overdoses are rare but their clinical 33 management is difficult, with poor efficacy of naloxone as antidote [56]. This is consistent 34 with the slow reversibility of 11C-buprenorphine binding by high-dose naloxone (0.22 mg/kg) 35 observed in our study. Previous blocking experiments performed in macaques and using the

13 1 µ-OR-selective radioligand 11C-carfentanil showed that ~85% occupancy of µ-OR was 2 achieved by a lower dose of naloxone (0.03 mg/kg, i.v., 10 min before PET) [38].

3 The occipital cortex and cerebellum are not proper reference tissue for 11C-buprenorphine 4 because of low but significant specific binding was found in these regions. We nonetheless 5 evaluated the occipital cortex as a pseudo-reference tissue region to non-invasively estimate 11 6 C-buprenorphine binding. Both DVRmicrodose and DVR0.003 mg/kg similarly predicted microdose 11 7 BPp (Fig. 4). In the absence of arterial input function, the binding potential of C- 8 buprenorphine in brain regions, which mainly reflects baseline availability of µ-OR, can 9 therefore be estimated using either microdose or low-dose 11C-buprenorphine 10 pharmacokinetic imaging using this simplified method.

11 For safety reasons, PET imaging is usually performed using microdose receptor antagonists 12 and low injected mass to avoid any adverse effects. In radiotracer development, co-injection 13 of radiotracers with pharmacological doses of corresponding unlabeled compounds is only 14 used to investigate the specific binding to brain regions [57]. We assume this strategy will 15 gain interest for multimodal pharmacological imaging protocols on simultaneous hybrid PET- 16 MR systems [58]. Using CNS-active dose, the time-course of PET-derived target 17 engagement can therefore be directly compared with the hemodynamic response assessed 18 using pharmacological MRI (phMRI) or other pharmacodynamic parameters in the same 19 individual [59]. Interestingly, the CNS effects of investigated doses of buprenorphine have 20 been studied using phMRI in both monkeys (0.03 mg/kg) [60] and humans (0.003 mg/kg) 21 [42]. In rhesus monkeys, buprenorphine increased the cerebral blood volume in brain regions 22 consistent with the binding of corresponding doses of buprenorphine to brain regions found 23 in our study [60].

24 Conclusion

25 Pharmacokinetic imaging provides a pragmatic method to explore the neuropharmacokinetic 26 and the µ-OR correlates of the CNS effects of buprenorphine. 11C-buprenorphine co-injected 27 with low dose buprenorphine could be safely performed as a dual-modality imaging 28 biomarker for PET/phMRI studies. This strategy may be useful to explore variability in 29 neurovascular coupling associated with the acute response to buprenorphine in future 30 multimodal pharmacological studies.

31

14 1 Funding and Disclosure

2 This work was performed on a platform member of France Life Imaging network (grant ANR- 3 11-INBS-0006) and was funded by the "Lidex-PIM" project funded by the IDEX Paris-Saclay, 4 ANR-11-IDEX-0003-02. There is no conflict of interest to disclose.

5

6 Acknowledgements

7 We gratefully thank Jérôme Cayla, Vincent Brulon and Maud Goislard for technical 8 assistance.

9

10 Author contribution

11 SA, MB, DV and NT contributed to conception of the work, data analysis and manuscript 12 writing. SA, SG and AP contributed to data acquisition. CW, MT and CL helped for PET and 13 MR imaging analysis. FC performed radiochemistry.

14

15 Supplementary information

16 Supplemental material accompanies this paper at (https://doi.org/).

17

15 1 References

2 1. Trescot AM, Datta S, Lee M, Hansen H. Opioid pharmacology. Pain Physician. 3 2008;11:S133-153. 4 2. Helm S, Trescot AM, Colson J, Sehgal N, Silverman S. Opioid antagonists, partial 5 agonists, and agonists/antagonists: the role of office-based detoxification. Pain 6 Physician. 2008;11:225–235. 7 3. Wakeman SE, Barnett ML. Primary Care and the Opioid-Overdose Crisis - 8 Buprenorphine Myths and Realities. N Engl J Med. 2018;379:1–4. 9 4. Olson KM, Duron DI, Womer D, Fell R, Streicher JM. Comprehensive molecular 10 pharmacology screening reveals potential new receptor interactions for clinically 11 relevant opioids. PloS One. 2019;14:e0217371. 12 5. Volpe DA, McMahon Tobin GA, Mellon RD, Katki AG, Parker RJ, Colatsky T, et al. 13 Uniform assessment and ranking of opioid μ receptor binding constants for selected 14 opioid drugs. Regul Toxicol Pharmacol. 2011;59:385–390. 15 6. Virk MS, Arttamangkul S, Birdsong WT, Williams JT. Buprenorphine is a weak partial 16 agonist that inhibits opioid receptor desensitization. J Neurosci. 2009;29:7341–7348. 17 7. Boas RA, Villiger JW. Clinical actions of fentanyl and buprenorphine. The significance of 18 receptor binding. Br J Anaesth. 1985;57:192–196. 19 8. Bidlack JM, Knapp BI, Deaver DR, Plotnikava M, Arnelle D, Wonsey AM, et al. In Vitro 20 Pharmacological Characterization of Buprenorphine, Samidorphan, and Combinations 21 Being Developed as an Adjunctive Treatment of Major Depressive Disorder. J 22 Pharmacol Exp Ther. 2018;367:267–281. 23 9. Lutfy K, Cowan A. Buprenorphine: a unique drug with complex pharmacology. Curr 24 Neuropharmacol. 2004;2:395–402. 25 10. Cami-Kobeci G, Polgar WE, Khroyan TV, Toll L, Husbands SM. Structural determinants 26 of opioid and NOP receptor activity in derivatives of buprenorphine. J Med Chem. 27 2011;54:6531–6537. 28 11. Raffa RB, Haidery M, Huang H-M, Kalladeen K, Lockstein DE, Ono H, et al. The clinical 29 analgesic efficacy of buprenorphine. J Clin Pharm Ther. 2014;39:577–583. 30 12. Walsh SL, Preston KL, Stitzer ML, Cone EJ, Bigelow GE. Clinical pharmacology of 31 buprenorphine: ceiling effects at high doses. Clin Pharmacol Ther. 1994;55:569–580. 32 13. Calabrese EJ. Pain and u-shaped dose responses: occurrence, mechanisms, and 33 clinical implications. Crit Rev Toxicol. 2008;38:579–590. 34 14. Bullingham RE, McQuay HJ, Moore A, Bennett MR. Buprenorphine kinetics. Clin 35 Pharmacol Ther. 1980;28:667–672. 36 15. Auvity S, Breuil L, Goislard M, Bottlaender M, Kuhnast B, Tournier N, et al. An original 37 radio-biomimetic approach to synthesize radiometabolites for PET imaging. Nucl Med 38 Biol. 2020:S0969805120301888. 39 16. Suridjan I, Comley RA, Rabiner EA. The application of positron emission tomography 40 (PET) imaging in CNS drug development. Brain Imaging Behav. 2019;13:354–365. 41 17. Nummenmaa L, Karjalainen T, Isojärvi J, Kantonen T, Tuisku J, Kaasinen V, et al. 42 Lowered endogenous mu-opioid receptor availability in subclinical depression and 43 anxiety. Neuropsychopharmacol. 2020. 30 May 2020. https://doi.org/10.1038/s41386- 44 020-0725-9. 45 18. Zubieta J, Greenwald MK, Lombardi U, Woods JH, Kilbourn MR, Jewett DM, et al. 46 Buprenorphine-induced changes in mu-opioid receptor availability in male heroin- 47 dependent volunteers: a preliminary study. Neuropsychopharmacol. 2000;23:326–334. 48 19. Greenwald MK, Johanson C-E, Moody DE, Woods JH, Kilbourn MR, Koeppe RA, et al. 49 Effects of buprenorphine maintenance dose on mu-opioid receptor availability, plasma 50 concentrations, and antagonist blockade in heroin-dependent volunteers. 51 Neuropsychopharmacol. 2003;28:2000–2009. 52 20. Greenwald MK, Comer SD, Fiellin DA. Buprenorphine maintenance and mu-opioid 53 receptor availability in the treatment of opioid use disorder: implications for clinical use 54 and policy. Drug Alcohol Depend. 2014;144:1–11.

16 1 21. Lever JR, Mazza SM, Dannals RF, Ravert HT, Wilson AA, Wagner HN. Facile synthesis 2 of [11C]buprenorphine for positron emission tomographic studies of opioid receptors. Int 3 J Rad Appl Instrum [A]. 1990;41:745–752. 4 22. Tournier N, Stieger B, Langer O. Imaging techniques to study drug transporter function 5 in vivo. Pharmacol Ther. 2018;189:104–122. 6 23. Ott J, Spilhaug MM, Maschauer S, Rafique W, Jakobsson JE, Hartvig K, et al. 7 Pharmacological Characterization of Low-to-Moderate Affinity Opioid Receptor Agonists 8 and Brain Imaging with 18F-Labeled Derivatives in Rats. J Med Chem. 2020;63:9484– 9 9499. 10 24. Chevillard L, Mégarbane B, Risède P, Baud FJ. Characteristics and comparative 11 severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and 12 buprenorphine in rats. Toxicol Lett. 2009;191:327–340. 13 25. Placzek MS, Schroeder FA, Che T, Wey H-Y, Neelamegam R, Wang C, et al. 14 Discrepancies in Kappa Opioid Agonist Binding Revealed through PET Imaging. ACS 15 Chem Neurosci. 2019;10:384–395. 16 26. Poisnel G, Oueslati F, Dhilly M, Delamare J, Perrio C, Debruyne D, et al. [11C]- 17 MeJDTic: a novel radioligand for kappa-opioid receptor positron emission tomography 18 imaging. Nucl Med Biol. 2008;35:561–569. 19 27. Auvity S, Chapy H, Goutal S, Caillé F, Hosten B, Smirnova M, et al. Diphenhydramine 20 as a selective probe to study H+-antiporter function at the blood-brain barrier: 21 Application to [11C]diphenhydramine positron emission tomography imaging. J Cereb 22 Blood Flow Metab. 2017;37:2185–2195. 23 28. Tempel A, Zukin RS. Neuroanatomical patterns of the mu, delta, and kappa opioid 24 receptors of rat brain as determined by quantitative in vitro autoradiography. Proc Natl 25 Acad Sci. 1987;84:4308–4312. 26 29. Tournier N, Goutal S, Auvity S, Traxl A, Mairinger S, Wanek T, et al. Strategies to Inhibit 27 ABCB1- and ABCG2-Mediated Efflux Transport of Erlotinib at the Blood-Brain Barrier: A 28 PET Study on Nonhuman Primates. J Nucl Med. 2017;58:117–122. 29 30. Auvity S, Tonietto M, Caillé F, Bodini B, Bottlaender M, Tournier N, et al. Repurposing 30 radiotracers for myelin imaging: a study comparing 18F-florbetaben, 18F-florbetapir, 31 18F-flutemetamol,11C-MeDAS, and 11C-PiB. Eur J Nucl Med Mol Imaging. 32 2020;47:490–501. 33 31. Reilhac A, Charil A, Wimberley C, Angelis G, Hamze H, Callaghan P, et al. 4D PET 34 iterative deconvolution with spatiotemporal regularization for quantitative dynamic PET 35 imaging. NeuroImage. 2015;118:484–493. 36 32. Rohlfing T, Kroenke CD, Sullivan EV, Dubach MF, Bowden DM, Grant KA, et al. The 37 INIA19 Template and NeuroMaps Atlas for Primate Brain Image Parcellation and 38 Spatial Normalization. Front Neuroinformatics. 2012;6:27. 39 33. Bentourkia M. Determination of the Input Function at the Entry of the Tissue of Interest 40 and Its Impact on PET Kinetic Modeling Parameters. Mol Imaging Biol. 2015;17:748– 41 756. 42 34. Logan J, Fowler JS, Volkow ND, Wolf AP, Dewey SL, Schlyer DJ, et al. Graphical 43 analysis of reversible radioligand binding from time-activity measurements applied to 44 [N-11C-methyl]-(-)-cocaine PET studies in human subjects. J Cereb Blood Flow Metab. 45 1990;10:740–747. 46 35. Martinez D, Hwang D, Mawlawi O, Slifstein M, Kent J, Simpson N, et al. Differential 47 occupancy of somatodendritic and postsynaptic 5HT(1A) receptors by pindolol: a dose- 48 occupancy study with [11C]WAY 100635 and positron emission tomography in humans. 49 Neuropsychopharmacol. 2001;24:209–229. 50 36. Logan J, Fowler JS, Volkow ND, Wang GJ, Ding YS, Alexoff DL. Distribution volume 51 ratios without blood sampling from graphical analysis of PET data. J Cereb Blood Flow 52 Metab. 1996;16:834–840. 53 37. Frost JJ, Douglass KH, Mayberg HS, Dannals RF, Links JM, Wilson AA, et al. 54 Multicompartmental analysis of [11C]-carfentanil binding to opiate receptors in humans

17 1 measured by positron emission tomography. J Cereb Blood Flow Metab. 1989;9:398– 2 409. 3 38. Saccone PA, Lindsey AM, Koeppe RA, Zelenock KA, Shao X, Sherman P, et al. 4 Intranasal Opioid Administration in Rhesus Monkeys: PET Imaging and Antinociception. 5 J Pharmacol Exp Ther. 2016;359:366–373. 6 39. Boyer EW. Management of opioid analgesic overdose. N Engl J Med. 2012;367:146– 7 155. 8 40. Cunningham VJ, Rabiner EA, Slifstein M, Laruelle M, Gunn RN. Measuring drug 9 occupancy in the absence of a reference region: the Lassen plot re-visited. J Cereb 10 Blood Flow Metab. 2010;30:46–50. 11 41. Henriksen G, Willoch F. Imaging of opioid receptors in the central nervous system. 12 Brain. 2008;131:1171–1196. 13 42. Upadhyay J, Anderson J, Schwarz AJ, Coimbra A, Baumgartner R, Pendse G, et al. 14 Imaging drugs with and without clinical analgesic efficacy. Neuropsychopharmacol. 15 2011;36:2659–2673. 16 43. Jenkins BG. Pharmacologic magnetic resonance imaging (phMRI): Imaging drug action 17 in the brain. NeuroImage. 2012;62:1072–1085. 18 44. Ide S, Minami M, Satoh M, Uhl GR, Sora I, Ikeda K. Buprenorphine antinociception is 19 abolished, but naloxone-sensitive reward is retained, in mu-opioid receptor knockout 20 mice. Neuropsychopharmacol. 2004;29:1656–1663. 21 45. Villiger JW, Taylor KM. Buprenorphine : characteristics of binding sites in the rat central 22 nervous system. Life Sci. 1981;29:2699–2708. 23 46. Frost JJ, Mayberg HS, Sadzot B, Dannals RF, Lever JR, Ravert HT, et al. Comparison 24 of [11C]diprenorphine and [11C]carfentanil binding to opiate receptors in humans by 25 positron emission tomography. J Cereb Blood Flow Metab. 1990;10:484–492. 26 47. Ragen BJ, Freeman SM, Laredo SA, Mendoza SP, Bales KL. μ and κ opioid receptor 27 distribution in the monogamous titi monkey (Callicebus cupreus): implications for social 28 behavior and endocrine functioning. Neuroscience. 2015;290:421–434. 29 48. Schadrack J, Willoch F, Platzer S, Bartenstein P, Mahal B, Dworzak D, et al. Opioid 30 receptors in the human cerebellum: evidence from [11C]diprenorphine PET, mRNA 31 expression and autoradiography. Neuroreport. 1999;10:619–624. 32 49. Hammers A, Lingford-Hughes A. Opioid imaging. Neuroimaging Clin N Am. 33 2006;16:529–552, vii. 34 50. Hiller JM, Fan LQ. Laminar distribution of the multiple opioid receptors in the human 35 cerebral cortex. Neurochem Res. 1996;21:1333–1345. 36 51. Valentino RJ, Volkow ND. Untangling the complexity of opioid receptor function. 37 Neuropsychopharmacol. 2018;43:2514–2520. 38 52. Hume SP, Lingford-Hughes AR, Nataf V, Hirani E, Ahmad R, Davies AN, et al. Low 39 sensitivity of the positron emission tomography ligand [11C]diprenorphine to agonist 40 opiates. J Pharmacol Exp Ther. 2007;322:661–667. 41 53. Gudin J, Fudin J. A Narrative Pharmacological Review of Buprenorphine: A Unique 42 Opioid for the Treatment of Chronic Pain. Pain Ther. 2020. 28 January 2020. 43 https://doi.org/10.1007/s40122-019-00143-6. 44 54. Bullingham RE, McQuay HJ, Porter EJ, Allen MC, Moore RA. Sublingual buprenorphine 45 used postoperatively: ten hour plasma drug concentration analysis. Br J Clin 46 Pharmacol. 1982;13:665–673. 47 55. Schottenfeld RS, Pakes J, O’Connor P, Chawarski M, Oliveto A, Kosten TR. Thrice- 48 weekly versus daily buprenorphine maintenance. Biol Psychiatry. 2000;47:1072–1079. 49 56. van Dorp E, Yassen A, Sarton E, Romberg R, Olofsen E, Teppema L, et al. Naloxone 50 reversal of buprenorphine-induced respiratory depression. Anesthesiology. 51 2006;105:51–57. 52 57. Pike VW. Considerations in the Development of Reversibly Binding PET Radioligands 53 for Brain Imaging. Curr Med Chem. 2016;23:1818–1869. 54 58. Tournier N, Comtat C, Lebon V, Gennisson J-L. Challenges and Perspectives of the 55 Hybridization of PET with Functional MRI or Ultrasound for Neuroimaging.

18 1 Neuroscience. 2020. 20 October 2020. 2 https://doi.org/10.1016/j.neuroscience.2020.10.015. 3 59. Sander CY, Hansen HD, Wey H-Y. Advances in simultaneous PET/MR for imaging 4 neuroreceptor function. J Cereb Blood Flow Metab. 2020:271678X20910038. 5 60. Seah S, Asad ABA, Baumgartner R, Feng D, Williams DS, Manigbas E, et al. 6 Investigation of cross-species translatability of pharmacological MRI in awake 7 nonhuman primate - a buprenorphine challenge study. PloS One. 2014;9:e110432. 8

9

19 1 Figures legends

2

3 Fig. 1. Impact of selected opioid antagonists on the regional binding of 11C- 4 buprenorphine in vivo in rats. PET acquisitions were performed without (baseline) or after 5 pharmacological blocking conditions using the non-selective OR antagonist naloxone (1 6 mg/kg i.v., 5 min before PET), the selective µ-OR antagonist naloxonazine (10 mg/kg i.v., 5 7 min before PET), the selective κ-OR antagonist norbinaltorphimine (10 mg/kg i.v., 30 min 8 before PET) and the selective δ-OR antagonist natrindole (3 mg/kg i.v., 5 min before PET). 9 Representative summed PET images (40-60min) obtained in each condition and 10 coregistered to a rat brain template are shown in A. Uptake ratios (region/cerebellum, 11 mean±S.D, n=4) are shown in B. ***p<0.001 compared with baseline, ns = non-significant.

12

13 Fig. 2. Parametric PET data of 11C-buprenorphine obtained from the co-injection study

14 in macaques. Representative parametric images expressed in VT (A). Regional VT measured 15 using the Logan plot analysis for each investigated brain region and each co-injected dose of 16 unlabeled buprenorphine (B). Data are shown as mean±SD, n=4). ***p<0.001 compared with 17 microdose, ns = non-significant.

18

11 19 Fig. 3. Correlation between C-buprenorphine VT measured during the co-injection 11 20 study and the corresponding microdose C-buprenorphine VT in macaques. Data are 21 represented as mean±SD. The slope of each correlation is indicated in the right panel.

22

23 Fig. 4. Correlation of outcome parameters derived from the kinetic modeling of 11C- 24 buprenorphine PET data obtained in macaques using microdose or therapeutic dose

25 of buprenorphine. Correlation between the binding potential (BPp;microdose) and the total 11 26 volume of distribution (VT;microdose) of microdose C-buprenorphine is shown in A. Correlation 27 of 11C-buprenorphine distribution volume ratio (DVR, Logan reference method) estimated in

28 microdose experiments (DVRmicrodose) and DVR obtained after co-injection with unlabeled

29 buprenorphine (DVR0.003mg/kg) is reported in B. Difference between DVRmicrodose and

30 DVR0.003mg/kg was not significant (paired t-test). Correlation of either DVRmicrodose or

31 DVR0.003mg/kg with BPp;microdose are shown in C and D, respectively. The coefficient of 32 determination (R2) is reported for the correlation of outcome parameters estimated in brain 33 regions of each individual.

20 Tables

Table 1. Outcome parameters obtained with PET pharmacokinetic modeling and in vivo binding experiments in macaques.

Estimated receptor occupancy (%) associated with Brain region VND,saturation VND,graphical BPp,microdose DVRmicrodo DVR0.003 mg/kg plasma levels of buprenorphine EC50 (µg/L) (= VT, 0.11 se 0.3µg/L 0.6µg/L 1 µg/L 3 µg/L 6 µg/L 9 µg/L mg/kg) Frontal cortex 3.62 ± 0.68 3.44 ± 1.70 4.80 ± 0.75 1.40 ± 0.19 1.47 ± 0.11 33.2 49.7 62.1 82.7 90.2 93.1 0.60 (0.07 to 1.12) Orbital cortex 3.56 ± 0.78 3.42 ± 1.33 4.01 ± 0.66 1.22 ± 0.15 1.39 ± 0.09 27.4 43.1 55.8 79.1 88.3 91.9 0.79 (0.30 to 1.30) Cingulate 3.77 ± 0.70 3.60 ± 1.78 5.48 ± 1.03 1.50 ± 0.28 1.67 ± 0.19 29.4 45.5 58.1 80.6 89.3 92.6 0.72 (0.33 to cortex 1.11) Temporal 3.67 ± 0.75 3.46 ± 2.13 3.88 ± 0.83 1.26 ± 0.15 1.41 ± 0.05 31.0 47.4 60.0 81.8 90 93.1 0.67 (0.18 to cortex 1.15) Parietal cortex 3.57 ± 0.63 3.41 ± 1.80 4.48 ± 1.05 1.37 ± 0.13 1.40 ± 0.10 34.3 51.1 63.5 83.9 91.3 94 0.58 (0.20 to 0.95) Occipital cortex 2.93 ± 0.62 2.84 ± 2.29 2.42 ± 1.05 NA NA † † † † † † †

Caudate 4.11 ± 0.56 3.91 ± 1.80 5.82 ± 1.39 1.57 ± 0.31 1.8 ± 0.25 23.6 38.3 50.8 75.6 86.1 90.3 0.99 (0.43 to 1.50) Putamen 4.42 ± 0.79 4.21 ± 1.83 6.41 ± 1.44 1.71 ± 0.31 1.9 ± 0.21 28.0 43.8 56.5 79.6 88.6 92.1 0.77 (0.34 to 1.20) Amygdala 3.92 ± 0.59 † 4.90 ± 1.05 1.39 ± 0.24 1.62 ± 0.19 14.5 25.3 36.1 62.9 77.2 83.6 1.77 (0.60 to 2.94) Thalamus 4.61 ± 0.76 4.33 ± 1.99 5.17 ± 0.93 1.54 ± 0.27 1.77 ± 0.19 23.4 37.9 50.4 75.3 85.9 90.1 0.98 (0.33 to 1.64) Hypothalamus 3.85 ± 0.70 † 4.38 ±0.79 1.27 ± 0.17 1.55 ± 0.08 23.4 37.9 50.4 75.3 85.9 90.1 0.98 (0.22 to 1.74) Cerebellum 3.55 ± 0.70 3.43 ± 2.13 2.21 ± 0.93 1.06 ± 0.12 1.11 ± 0.05 † † † † † † †

VND is the non-displaceable volume of distribution. VND,saturation has been estimated using the Logan plot method and the maximum co-injected dose of unlabeled buprenorphine (VT, 0.11 mg/kg). VND,graphical has been graphically estimated (see supplemental material, Fig. S3). BPp is the binding potential relative to the plasma kinetic of 11C-buprenorphine. DVR is the distribution volume ratio estimated with the Logan reference method using the occipital cortex as the pseudo-reference region. EC50 is the estimated plasma concentration of buprenorphine associated to

21 50% of buprenorphine brain receptor occupancy. NA = non-applicable, † = poorly estimated. Data are expressed as mean (receptor occupancy) or mean ± SD. Estimated EC50 are reported as mean (confidence interval 95%).

22 1 Figures

2

3 Figure. 1

A

3 Uptake ratio

Baseline Naloxonazine Naltrindole Norbinaltorphimine Naloxone 1

κ B Baseline Norbinaltorphimine ( -antagonist) Naloxonazine (μ-antagonist) Naloxone (μ-, δ-, κ-antagonist) Naltrindole (δ-antagonist)

5 ns ns ns ns ns 4 ns ns 3 ns ns ns ns ns

2 *** *** *** *** *** *** *** *** *** *** Uptake ratio *** *** 1 Region / Cerebellum / Region

0 4 Amygdala Striatum Hippocampus Thalamus Hypothalamus Cortex 5

23 1 Figure 2

2

3 4 5

24 1 Figure 3 2

15 OR-rich regions Condition Slope Microdose 1.00 10 Occipital Cortex 0.003 mg/kg 1.05 Cerebellum 0.006 mg/kg 0.82

T;dose 0.03 mg/kg V 0.47 5 0.06 mg/kg 0.27 0.11 mg/kg 0.22 0 4 6 8 10 12

VT;microdose 3 4 5

25 1 Figure 4

A B

15 2.5 2 ) R = 0.87 -3 Paired t-test P > 0.05 R2 = 0.98 2.0 10 (mL.cm 1.5 0.003 mg/kg 5

DVR 1.0 p; microdose p; BP 0 0.5 0 5 10 15 0.5 1.0 1.5 2.0 2.5 -3 DVR VT; Microdose (mL.cm ) microdose C D

10 10

) ) -3 -3 8 R2= 0.64 8 R2= 0.66 6

(mL.cm 6 (mL.cm

4 4 p , microdose p , p; microdose p; microdose 2 2 BP BP 0 0 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

DVRmicrodose DVR0.003 mg/kg 2

26